Visfatin induces MUC8 and MUC5B expression via p38 MAPK/ROS/NF-κB in human airway epithelial cells

Background

Among a variety of inflammatory mediators, visfatin is a proinflammatory adipocytokine associated with inflammatory reactions in obesity, metabolic syndrome, chronic inflammatory disease, and autoimmune disease. However, the biological role of visfatin in secretion of major mucins in human airway epithelial cells has not been reported. Therefore, this study was conducted in order to investigate the effect and the brief signaling pathway of visfatin on MUC8 and MUC5B expression in human airway epithelial cells.

Results

Visfatin significantly induced MUC8 and MUC5B expression. Visfatin significantly activated phosphorylation of p38 MAPK. Treatment with SB203580 (p38 MAPK inhibitor) and knockdown of p38 MAPK by siRNA significantly blocked visfatin-induced MUC8 and MUC5B expression.Visfatin significantly increased ROS formation. Treatment with SB203580 significantly attenuated visfatin-induced ROS formation. Treatment with NAC (ROS scavenger) and DPI (NADPH oxidase inhibitor) significantly attenuated visfatin-induced MUC8 and MUC5B expression. However, treatment with NAC and DPI did not attenuate visfatin-activated phosphorylation of p38 MAPK. Visfatin significantly activated the phosphorylation of NF-κB. Treatment with PDTC (NF-κB inhibitor) significantly attenuated visfatin-induced MUC8 and MUC5B expression.

Conclusions

These results suggest that visfatin induces MUC8 and MUC5B expression through p38 MAPK/ROS/NF-κB signaling pathway in human airway epithelial cells.     

MUC1 mediates Pneumocystis murina binding to airway epithelial cells

Previous studies have shown that Pneumocystis binds to pneumocytes, but the proteins responsible for binding have not been well defined. Mucins are the major glycoproteins present in mucus, which serves as the first line of defence during airway infection. MUC1 is the best characterised membrane‐tethered mucin and is expressed on the surface of most airway epithelial cells. Although by electron microscopy Pneumocystis primarily binds to type I pneumocytes, it can also bind to type II pneumocytes. We hypothesized that Pneumocystis organisms can bind to MUC1 expressed by type II pneumocytes. Overexpression of MUC1 in human embryonic kidney HEK293 cells increased Pneumocystis binding, while knockdown of MUC1 expression by siRNA in A549 cells, a human adenocarcinoma‐derived alveolar type II epithelial cell line, decreased Pneumocystis binding. Immunofluorescence labelling indicated that MUC1 and Pneumocystis were co‐localised in infected mouse lung tissue. Incubation of A549 cells with Pneumocystis led to phosphorylation of ERK1/2 that increased with knockdown of MUC1 expression by siRNA. Pneumocystis caused increased IL‐6 and IL‐8 secretion by A549 cells, and knockdown of MUC1 further increased their secretion in A549 cells. Taken together, these results suggest that binding of Pneumocystis to MUC1 expressed by airway epithelial cells may facilitate establishment of productive infection.     

LPS stimulates MUC5AC expression in human biliary epithelial cells: whether there exists a possible pathway of PKC/NADPH/ROS?

Previous studies have shown that lipopolysaccharide (LPS) can upregulate MUC5AC in airway epithelial cells. However, the relationship and mechanism between bacterial infection and altered mucus secretion in the biliary tract remains unclear. Human biliary epithelial cells were induced by LPS, H₂O₂ production in the cell supernatants were detected by specific kit and expression of MUC5AC were detected by real-time PCR, Western blot, and immunohistochemistry. H₂O₂ production increased in a dose-dependent manner, LPS upregulate MUC5AC expression in both mRNA and protein level while specific inhibitors can reduce this high expression. Reactive oxygen species participates in the process of LPS by upregulating MUC5AC secretion. Moreover, PKC and NADPH oxidase regulate MUC5AC production in LPS-challenged human biliary epithelial cells.     

Coordinate Regulation of the Gel-forming Mucin Genes at Chromosome 11p15.5

Four of the genes that encode gel-forming mucins, which are major components of the mucus layer protecting many epithelial surfaces, are clustered at chromosome 11p15.5 and show both cell- and tissue-specific expression patterns. We aimed to determine whether the individual genes were coordinately regulated by mechanisms involving higher order chromatin structure. CCCTC-binding factor (CTCF) sites were predicted in silico and CTCF occupancy then evaluated by chromatin immunoprecipitation. CTCF was found at many sites across the gene cluster, and its binding was correlated with mucin gene expression. Next, siRNA-mediated depletion of CTCF was shown to increase MUC2 expression in A549 lung carcinoma cells and both MUC6 and MUC5AC expression in LS180 colon carcinoma cells. These changes correlated with loss of CTCF binding at multiple sites, although others retained occupancy. In cells actively expressing the mucins, the gene cluster was shown by chromosome conformation capture to form looped three-dimensional structures with direct interactions between the MUC2 promoter region, regions 30 kb 5′ to it, close to the MUC6 promoter and others near the 3′ end of MUC5AC, >170 kb away. Finally, to demonstrate the importance of CTCF binding to mucin gene expression, Calu-3 lung carcinoma cells were exposed to lipopolysaccharide (LPS). LPS increased the expression of MUC2 and MUC5AC and reduced MUC5B. CTCF occupancy was concurrently depleted at specific binding sites close to these genes. These data suggest that CTCF binding and cell type-specific long-range interactions across the 11p15.5 gene cluster are critical mechanisms for coordinating gel-forming mucin gene expression.     

Expression of MUC1 mucin in human umbilical vein endothelial cells (HUVEC)

Mucin 1 (MUC1) is a membrane-bound glycoprotein that is expressed by various epithelial cell types. MUC1 functions include modulation of cell adhesion, signal transduction, lubrication and hydration of epithelial surfaces, and their protection from infection. In this study we demonstrated that MUC1 is expressed in human umbilical vein endothelial cells (HUVECs) and could be released/shed from cellular membrane. MUC1 presence in these cells was verified using three methods: Western blotting, flow cytometry and metabolic labeling. We also showed that mucin expression is stimulated by proinflammatory cytokines: about a 2-fold increase was observed after TNF-α treatment and lower after IFN-γ alone and in combination with TNF-α treatment. It can be assumed that the presence of MUC1 in endothelial cells may have an important role in the interactions with different cell types in physiological and pathological processes.     

Protein expression by Streptococcus uberis in co-culture with bovine mammary epithelial cells

Three strains of Streptococcus uberis isolated from dairy cows with mastitis were co-cultured with a bovine mammary epithelial cell line (MAC-T) in Dulbecco's modified Eagle's medium without fetal bovine serum. Protein profiles from culture supernatants and bacterial pellets among different treatments were compared by electrophoresis. There were proteins induced or having increased expression in both supernatant and surface-associated samples from S. uberis co-cultured with MAC-T cells. Some of these proteins were recognized by antibodies in serum obtained from a cow infected by S. uberis . In supernatant samples, there were two distinct protein bands at 35 and 36.8 kDa for all three strains of S. uberis co-cultured with MAC-T cells. These two bands were absent when bacterial protein synthesis was inhibited by chloramphenicol. This study clearly indicates that bacterial protein expression was regulated in response to co-culture with mammary epithelial cells.     

A1 adenosine receptor signaling reduces Streptococcus pneumoniae adherence to pulmonary epithelial cells by targeting expression of platelet‐activating factor receptor

Extracellular adenosine production is crucial for host resistance against Streptococcus pneumoniae (pneumococcus) and is thought to affect antibacterial immune responses by neutrophils. However, whether extracellular adenosine alters direct host–pathogen interaction remains unexplored. An important determinant for lung infection by S. pneumoniae is its ability to adhere to the pulmonary epithelium. Here we explored whether extracellular adenosine can directly impact bacterial adherence to lung epithelial cells. We found that signaling via A1 adenosine receptor significantly reduced the ability of pneumococci to bind human pulmonary epithelial cells. A1 receptor signaling blocked bacterial binding by reducing the expression of platelet‐activating factor receptor, a host protein used by S. pneumoniae to adhere to host cells. In vivo, A1 was required for control of pneumococcal pneumonia as inhibiting it resulted in increased host susceptibility. As S. pneumoniae remain a leading cause of community‐acquired pneumonia in the elderly, we explored the role of A1 in the age‐driven susceptibility to infection. We found no difference in A1 pulmonary expression in young versus old mice. Strikingly, triggering A1 signaling boosted host resistance of old mice to S. pneumoniae pulmonary infection. This study demonstrates a novel mechanism by which extracellular adenosine modulates resistance to lung infection by targeting bacterial–host interactions.     

Role of glutathione in augmenting the anticancer activity of pyrroloquinoline quinone (PQQ)

Abstract

Pyrroloquinoline quinone (PQQ), a bacterial redox co-factor and antioxidant, is highly reactive with nucleophilic compounds present in biological fluids. PQQ induced apoptosis in human promonocytic leukemia U937 cells and this was accompanied by depletion of the major cellular antioxidant glutathione and increase in intracellular reactive oxygen species (ROS). Treatment with glutathione (GSH) or N-acetyl-L-cysteine (NAC) did not spare PQQ toxicity but resulted in a 2–5-fold increase in PQQ-induced apoptosis in U937 cells. Cellular GSH levels increased following treatment by NAC alone but were severely depleted by co-treatment with NAC and PQQ. This was accompanied by an increase in intracellular ROS. Alternatively, depletion of glutathione also resulted in increased PQQ cytotoxicity. However, the cells underwent necrosis as evidenced by dual labeling with annexin V and propidium iodide. PQQ-induced cytotoxicity is thus critically regulated by the cellular redox status. An increase in GSH can augment apoptosis and its depletion can switch the mode of cell death to necrosis in the presence of PQQ. Our data suggest that modulation of intracellular GSH can be used as an effective strategy to potentiate cytotoxicity of quinones like PQQ.     

Menthol induces cell-cycle arrest in PC-3 cells by down-regulating G2/M genes, including polo-like kinase 1

High-mobility group box-1 protein (HMGB1), which is produced by immune cells, was recently identified as a proinflammatory mediator in various inflammatory diseases. In this study, we investigated the effect of HMGB1 on the expression of mucin (MUC) genes in human airway epithelial cells. We showed that HMGB1 markedly increased MUC8 expression, and that the expression of other MUC genes was also regulated by HMGB1. HMGB1 activated the JNK and PI3K/Akt signaling pathways, and inhibitors of JNK and PI3K/Akt markedly inhibited HMGB1-induced MUC8 expression. Furthermore, HMGB1 increased the production of intracellular reactive oxygen species (ROS). However, the ROS scavengers Trolox and N-acetylcysteine (NAC) had no effect on MUC8 expression in HMGB1-treated NCI-H292 cells. Taken together, our results suggest that HMGB1 induces MUC8 expression in a JNK and PI3K/Akt signaling pathway-dependent manner but that HMGB1 acts in an ROS-independent manner.     

AP2α is essential for MUC8 gene expression in human airway epithelial cells

Mucins are high molecular weight proteins that make up the major components of mucus. Hypersecretion of mucus is a feature of several chronic inflammatory airway diseases. MUC8 is an important component of airway mucus, and its gene expression is upregulated in nasal polyp epithelium. Little is known about the molecular mechanisms of MUC8 gene expression. We first observed overexpression of activator protein‐2alpha (AP2α) in human nasal polyp epithelium. We hypothesized that AP2α overexpression in nasal polyp epithelium correlates closely with MUC8 gene expression. We demonstrated that phorbol 12‐myristate 13‐acetate (PMA) treatment of the airway epithelial cell line NCI‐H292 increases MUC8 gene and AP2α expression. In this study, we sought to determine which signal pathway is involved in PMA‐induced MUC8 gene expression. The results show that the protein kinase C and mitogen‐activating protein/ERK kinase (MAPK) pathways modulate MUC8 gene expression. PD98059 or ERK1/2 siRNA and RO‐31‐8220 or PKC siRNA significantly suppress AP2α as well as MUC8 gene expression in PMA‐treated cells. To verify the role of AP2α, we specifically knocked down AP2α expression with siRNA. A significant AP2α knock‐down inhibited PMA‐induced MUC8 gene expression. While dominant negative AP2α decreased PMA‐induced MUC8 gene expression, overexpressing wildtype AP2α increased MUC8 gene expression. Furthermore, using lentiviral vectors for RNA interference in human nasal polyp epithelial cells, we confirmed an essential role for AP2α in MUC8 gene expression. From these results, we concluded that PMA induces MUC8 gene expression through a mechanism involving PKC, ERK1/2, and AP2α activation in human airway epithelial cells. J. Cell. Biochem. 110: 1386–1398, 2010. © 2010 Wiley‐Liss, Inc.     

Lymphocytes Accelerate Epithelial Tight Junction Assembly: Role of AMP-Activated Protein Kinase (AMPK)

The tight junctions (TJs), characteristically located at the apicolateral borders of adjacent epithelial cells, are required for the proper formation of epithelial cell polarity as well as for sustaining the mucosal barrier to the external environment. The observation that lymphocytes are recruited by epithelial cells to the sites of infection [1] suggests that they may play a role in the modulation of epithelial barrier function and thus contribute to host defense. To test the ability of lymphocytes to modulate tight junction assembly in epithelial cells, we set up a lymphocyte-epithelial cell co-culture system, in which Madin-Darby canine kidney (MDCK) cells, a well-established model cell line for studying epithelial TJ assembly [2], were co-cultured with mouse lymphocytes to mimic an infection state. In a typical calcium switch experiment, the TJ assembly in co-culture was found to be accelerated compared to that in MDCK cells alone. This accelaration was found to be mediated by AMP-activated protein kinase (AMPK). AMPK activation was independent of changes in cellular ATP levels but it was found to be activated by the pro-inflammatory cytokine TNF-α. Forced suppression of AMPK, either with a chemical inhibitor or by knockdown, abrogated the accelerating effect of lymphocytes on TJ formation. Similar results were also observed in a co-culture with lymphocytes and Calu-3 human airway epithelial cells, suggesting that the activation of AMPK may be a general mechanism underlying lymphocyte-accelerated TJ assembly in different epithelia. These results suggest that signals from lymphocytes, such as cytokines, facilitate TJ assembly in epithelial cells via the activation of AMPK.     

The effect of N‐acetylcysteine on chloride efflux from airway epithelial cells

Defective chloride transport in epithelial cells increases mucus viscosity and leads to recurrent infections with high oxidative stress in patients with CF (cystic fibrosis). NAC (N‐acetylcysteine) is a well known mucolytic and antioxidant drug, and an indirect precursor of glutathione. Since GSNO (S‐nitrosoglutathione) previously has been shown to be able to promote Cl^? efflux from CF airway epithelial cells, it was investigated whether NAC also could stimulate Cl^? efflux from CF and non‐CF epithelial cells and through which mechanisms. CFBE (CF bronchial epithelial cells) and normal bronchial epithelial cells (16HBE) were treated with 1 mM, 5 mM, 10 mM or 15 mM NAC for 4 h at 37°C. The effect of NAC on Cl^? transport was measured by Cl^? efflux measurements and by X‐ray microanalysis. Cl^? efflux from CFBE cells was stimulated by NAC in a dose‐dependent manner, with 10 mM NAC causing a significant increase in Cl^? efflux with nearly 80% in CFBE cells. The intracellular Cl^? concentration in CFBE cells was significantly decreased up to 60% after 4 h treatment with 10 mM NAC. Moreover immunocytochemistry and Western blot experiments revealed expression of CFTR channel on CFBE cells after treatment with 10 mM NAC. The stimulation of Cl^? efflux by NAC in CF airway epithelial cells may improve hydration of the mucus and thereby be beneficial for CF patients.     

Altered expression of inflammatory cytokine receptors in response to LPS challenge through interaction between intestinal epithelial cells and lymphocytes of Peyer's patch

The intense innate immunological activities occurring at the enteric mucosal surface involve interactions between intestinal epithelial cells and immune cells. Our previous studies have indicated that Peyer's patch lymphocytes may modulate intestinal epithelial barrier and ion transport function in homeostasis and host defense via cell-cell contact as well as cytokine signaling. The present study was undertaken using the established co-culture system of Caco-2 epithelial cells with lymphocytes of Peyer's patch to investigate the expression of IL-8 and IL-6 cytokines and cytokine receptors in the co-culture system after challenge with Shigella F2a-12 lipopolysaccharide (LPS). The human colonic epithelial cell line Caco-2 was co-cultured with freshly isolated lymphocytes from the murine Peyer's patch either in the mixed or separated (isolated but permeable compartments) co-culture configuration, and was challenged with Shigella F2a-12 LPS for 8 h. The level of mRNA expressions of human interleukin-8 (hIL-8), human interleukin-8 receptor (hIL-8R), mouse interleukin-8 receptor (mIL-8R), mouse interleukin-6 (mIL-6), mouse interleukin-6 receptor (mIL-6R) and human interleukin-6 receptor (hIL-6R) was examined by semi-quantitative PCR. In both co-culture groups, hIL-8 expression of Caco-2 cells was decreased, and hIL-8R expression was increased compared to the Caco-2 alone group. Upon LPS challenge, hIL-8 expression from the Caco-2 cells of both co-culture groups was higher than in the Caco-2 control group. The increased hIL-8 expression of Caco-2 cells in the separated co-culture group is correlated with a decreased hIL-8R expression and an increased mIL-8R expression. In the mixed co-culture group, the increased expression of hIL-8 was associated with the upregulated hIL-8R expression on Caco-2 cells and downregulated mIL-8R on murine Peyer's patch lymphocytes (PPL). mIL-6 expression from mouse PPL was also upregulated by LPS in mixed co-culture. However, upon the treatment with LPS, hIL-6R expression of Caco-2 cells was decreased in the mixed co-culture, but increased in separated co-culture. The data suggest that release of hIL-8 from epithelial cells may act on lymphocytes through a paracrine pathway, but it may also act on the epithelial cells themselves via an autocrine pathway. The data also suggest that the release of mIL-6 from Peyer's patch lymphocytes affects epithelial cells in a paracrine fashion.     

Tropism of and Innate Immune Responses to the Novel Human Betacoronavirus Lineage C Virus in Human Ex Vivo Respiratory Organ Cultures

Since April 2012, there have been 17 laboratory-confirmed human cases of respiratory disease associated with newly recognized human betacoronavirus lineage C virus EMC (HCoV-EMC), and 7 of them were fatal. The transmissibility and pathogenesis of HCoV-EMC remain poorly understood, and elucidating its cellular tropism in human respiratory tissues will provide mechanistic insights into the key cellular targets for virus propagation and spread. We utilized ex vivo cultures of human bronchial and lung tissue specimens to investigate the tissue tropism and virus replication kinetics following experimental infection with HCoV-EMC compared with those following infection with human coronavirus 229E (HCoV-229E) and severe acute respiratory syndrome coronavirus (SARS-CoV). The innate immune responses elicited by HCoV-EMC were also investigated. HCoV-EMC productively replicated in human bronchial and lung ex vivo organ cultures. While SARS-CoV productively replicated in lung tissue, replication in human bronchial tissue was limited. Immunohistochemistry revealed that HCoV-EMC infected nonciliated bronchial epithelium, bronchiolar epithelial cells, alveolar epithelial cells, and endothelial cells. Transmission electron microscopy showed virions within the cytoplasm of bronchial epithelial cells and budding virions from alveolar epithelial cells (type II). In contrast, there was minimal HCoV-229E infection in these tissues. HCoV-EMC failed to elicit strong type I or III interferon (IFN) or proinflammatory innate immune responses in ex vivo respiratory tissue cultures. Treatment of human lung tissue ex vivo organ cultures with type I IFNs (alpha and beta IFNs) at 1 h postinfection reduced the replication of HCoV-EMC, suggesting a potential therapeutic use of IFNs for treatment of human infection.     

Protein changes in gastric epithelial cells RGM-1 in response to Helicobacter pylori infection

Helicobacter pylori-induced inflammation significantly increases the risk of gastric cancer. To investigate the role of H. pylori infection in gastric epithelial cell carcinogenesis, flow cytometry was used to analyze the apoptosis of gastric epithelial cells infected by H. pylori. Next, LTQ MS mass spectrometry (MS) was applied to identify protein changes in gastric epithelial cells infected with H. pylori, and then bioinformatics was adopted to analyze the cellular localization and biological function of differential proteins. LTQ MS/MS successfully identified identified 22 differential proteins successfully, including 20 host-cell proteins and two H. pylori bacterial proteins. Also, human proteins were located in all areas of cells and involved in various cell biological functions. The oncogene proteins p53, p16, and C-erbB-2 proteins in H. pylori-infected RGM-1 cells were remarkably increased from the analysis by Western blot analysis. H. pylori infection of gastric epithelial cells leads to changes in various protein components in the cell, and enhances the expression of oncogene proteins, thereby increasing the possibility of possibility of carcinogenesis of H. pylori infection.     

TNF-alpha sensitizes HT-29 colonic epithelial cells to intestinal lactobacilli

The ability of the proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) to influence epithelial interleukin (IL)-8 responses to the intestinal bacterium Lactobacillus plantarum 299v was analyzed in the human HT-29 colonic epithelial cell line. In the absence of TNF-alpha, IL-8 mRNA expression was not detectable by Northern blot analysis in HT-29 cells alone or in HT-29 cells co-cultured with L. plantarum 299v. However, TNF-alpha induced IL-8 mRNA expression, and co-culture of TNF-alpha-treated HT-29 cells with L. plantarum 299v significantly increased IL-8 mRNA expression above levels induced by TNF-alpha alone in an adhesion-dependent manner. The increase in IL-8 mRNA expression was not observed in TNF-alpha-treated HT-29/L. plantarum 299v co-cultures using heat-killed lactobacilli or when L. plantarum adhesion was prevented using mannoside or a trans-well membrane. Paradoxically, IL-8 secretion was decreased in TNF-alpha-treated HT-29 cells with L. plantarum 299v relative to cells treated with TNF-alpha alone. TNF-alpha-mediated responsiveness to L. plantarum 299v was further investigated by analyzing expression of a coreceptor for bacterial cell wall products CD14. HT-29 cells expressed CD14 mRNA and cell-surface CD14; however, TNF-alpha did not alter CD14 mRNA or cell-surface expression, and blockade of CD14 with monoclonal antibody MY4 did not alter the IL-8 response to L. plantarum 299v in TNF-alpha-treated HT-29 cells. These results indicate that although TNF-alpha sensitizes HT-29 epithelial cells to intestinal lactobacilli, the bacteria exert a protective effect by downregulating IL-8 secretion.